DESCRIPTION (Verbatim from the applicant's abstract): The long range objective of this project is to identify proteins and characterize molecular mechanisms involved in organizing microtubule minus ends at spindle poles in mammalian cells. Accurate chromosome segregation is essential for the propagation of species and the viability of all cells. Chromosomes are segregated during mitosis and meiosis by a complex microtubule-based superstructure called the spindle. Microtubules within the spindle have their plus ends extending either toward the cell cortex or to the cell equator where they contact the chromosomes or interact with other microtubules. Microtubule minus ends are focused at two unique positions referred to as spindle poles. The spindle poles are the functional sites to which the sister chromatids will segregate during anaphase. Recent evidence has shown that microtubule minus ends are released and/or severed from the primary site of nucleation associated with the centrosomes. Following dissociation from the centrosome, a collection of structural and motor proteins work together to tightly focus microtubule minus ends at the spindle pole. We have devised a cell free assay that faithfully reproduces the centrosome-independent aspects of spindle pole organization. Combining this assay with in vivo approaches to monitor spindle organization provide my laboratory with a unique opportunity to characterize the proteins and mechanisms involved in spindle pole organization and function in mammalian cells. It is the goal of this project to identify proteins involved in focusing microtubule minus ends at spindle poles and to use both in vitro and in vivo biochemical experiments to dissect the molecular mechanisms involved in this essential process. The specific aims of this research are to: 1) determine the dynamics of the association of NuMA with microtubule minus ends at spindle poles; 2) characterize the functional properties of a large complex that contains the minus end-directed microtubule motor protein HSET; and 3) perform a molecular dissection of spindle pole components using microtubule asters assembled in a cell free mitotic extract.